Transcription factor Nrf2 activation regulates NETosis, endothelial injury, and kidney disease in myeloperoxidase-positive antineutrophil cytoplasmic antibody-associated vasculitis.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a systemic autoimmune disease pathologically characterized by vascular necrosis with inflammation. During AAV development, activated neutrophils produce reactive oxygen species (ROS), leading to the aberrant formation of neutrophil extracellular traps (NETs) via NETosis and subsequent fibrinoid vascular necrosis. Nuclear factor-erythroid 2-related factor 2 (Nrf2) functions as an intracellular defense system to counteract oxidative stress by providing antioxidant properties. Herein, we explored the role of Nrf2 in the pathogenesis of AAV. The role and mechanism of Nrf2 in ANCA-stimulated neutrophils and subsequent endothelial injury were evaluated in vitro using Nrf2 genetic deletion and Nrf2 activator treatment. In corresponding in vivo studies, the role of Nrf2 in ANCA-transfer AAV and spontaneous AAV murine models was examined. Pharmacological activation of Nrf2 in vitro suppressed ANCA-induced NET formation via the inhibition of ROS. In contrast, NET formation was enhanced in Nrf2-deficient neutrophils. Furthermore, Nrf2 activation protected endothelial cells from ANC-induced NETs-mediated injury. In vivo, Nrf2 activation ameliorated glomerulonephritis in two AAV models by upregulating antioxidants and inhibiting ROS-mediated NETs. Furthermore, Nrf2 activation restrained the expansion of splenic immune cells, including T lymphocytes and limited the infiltration of Th17 cells into the kidney. In contrast, Nrf2 genetic deficiency exacerbated vasculitis in a spontaneous AAV model. Thus, the pathophysiological process in AAV may be downregulated by Nrf2 activation, potentially leading to a new therapeutic strategy by regulating NETosis.

Utility of the refined EBMT diagnostic and severity criteria 2023 for sinusoidal obstruction syndrome/veno-occlusive disease.

Sinusoidal obstruction syndrome/veno-occlusive disease (SOS/VOD) is a life-threatening complication of hematopoietic stem cell transplantation (HSCT). Early diagnosis of SOS/VOD is associated with improved clinical outcomes. In 2023, the refined European Society for Blood and Marrow Transplantation diagnostic and severity criteria (refined EBMT criteria 2023) have been advocated. The revision has introduced new diagnostic categories, namely; probable, clinical, and proven SOS/VOD. In addition, the Sequential Organ Failure Assessment (SOFA) score has been newly incorporated into the SOS/VOD severity grading. We performed a retrospective analysis to evaluate the utility of these criteria. We analyzed 161 cases who underwent allogeneic HSCT. We identified 53 probable, 23 clinical, and 4 proven SOS/VOD cases. Probable SOS/VOD was diagnosed a median of 5.0 days earlier (interquartile range: 2-13 days, P < 0.001) than that of clinical SOS/VOD. The development of probable SOS/VOD alone was associated with a significantly inferior survival proportion compared to non-SOS/VOD (100-day survival, 86.2% vs. 94.3%, P = 0.012). The SOFA score contributed to the prediction of prognosis. Consequently, the refined EBMT criteria 2023 demonstrated the utility of SOS/VOD diagnosis and severity grading. Further investigations and improvements in these criteria are warranted.

Combination Therapy with EGFR Tyrosine Kinase Inhibitors and TEAD Inhibitor Increases Tumor Suppression Effects in EGFR Mutation-positive Lung Cancer.

EGFR-tyrosine kinase inhibitors (TKI) are the first-line therapies for EGFR mutation-positive lung cancer. EGFR-TKIs have favorable therapeutic effects. However, a large proportion of patients with EGFR mutation-positive lung cancer subsequently relapse. Some cancer cells survive the initial treatment with EGFR-TKIs, and this initial survival may be associated with subsequent recurrence. Therefore, we aimed to overcome the initial survival against EGFR-TKIs. We hypothesized that yes-associated protein 1 (YAP1) is involved in the initial survival against EGFR-TKIs, and we confirmed the combined effect of EGFR-TKIs and a YAP1-TEAD pathway inhibitor. The KTOR27 (EGFR kinase domain duplication) lung cancer cell lines established from a patient with EGFR mutation-positive lung cancer and commercially available PC-9 and HCC827 (EGFR exon 19 deletions) lung cancer cell lines were used. These cells were used to evaluate the in vitro and in vivo effects of VT104, a TEAD inhibitor. In addition, YAP1 involvement was investigated in pathologic specimens. YAP1 was activated by short-term EGFR-TKI treatment in EGFR mutation-positive lung cancer cells. In addition, inhibiting YAP1 function using siRNA increased the sensitivity to EGFR-TKIs. Combination therapy with VT104 and EGFR-TKIs showed better tumor-suppressive effects than EGFR-TKIs alone, in vitro and in vivo. Moreover, the combined effect of VT104 and EGFR-TKIs was observed regardless of the localization status of YAP1 before EGFR-TKI exposure. These results suggest that combination therapy with the TEAD inhibitor and EGFR-TKIs may improve the prognosis of patients with EGFR mutation-positive lung cancer.

[Inflammatory Polyp due to Peanut Aspiration, Needed to Differentiate from Malignant Tumor: Report of a Case].

The patient was in his 70s. He was addmitted to our hospital because of obstructive pneumonia for 3 months. Chest computed tomography( CT) showed a nodule at the base of the right B8, obstructing the basal branch, with consolidation of the peripheral lung. Bronchoscopy revealed the right basal trunk obstruction by a tumorous lesion. FDG-PET showed heterogeneous FDG uptake at the right hilum and the lower lobe suggesting malignancy, and a thoracoscopic right lower lobectomy was performed. Pathology showed a granulation-like nodule and a brown oval foreign body incarcerated in the peripheral bronchus, which was later revealed to be a peanut, and no obvious malignant findings were observed.

[Invasive Mucinous Adenocarcinoma that Invades the Wall of a Cystic Lesion:Report of a Case].

Invasive mucinous adenocarcinoma (IMA) is a rare and special type of lung adenocarcinoma. We report a case of IMA presenting as a cystic lesion in the S10 of the right lung, diagnosed by surgical biopsy and treated with right lower lobectomy. The patient was a 60-year-old man who was found to have a 10-mm-sized frosted ground-glass opacity with a 10-mm-sized air space in the S10 of the right lung while undergoing follow-up after renal cancer surgery in 2018. The air space gradually enlarged and, in 2022, began to show a 40-mm-sized cyst, with partial wall thickening and nodularity on the caudal side. A thoracoscopic partial pneumonectomy was performed to confirm the diagnosis of IMA, and a thoracoscopic radical resection of the right remaining lower lobe was performed. It is important to recognize that adenocarcinoma may occur in patients with thin-wall cavity, as in this case. Additionally, it is necessary to determine the treatment strategy based on the assumption that the tumor may extend to the entire cavity wall, even if it is thin-walled.

The risk of weekend biopsy: Impact of specimen source and fixation status on HER2 assessment in the treatment of advanced gastric cancer (The HER_WEEKEND study).

Accurate evaluation of human epidermal growth factor receptor type 2 (HER2) expression is crucial for determining chemotherapy regimens in gastric cancer. However, formalin fixation status has been identified as an important factor affecting HER2 assessment reliability. This retrospective cohort study aimed to investigate the correlation between sample collection day (weekday vs. weekend) and source (biopsy vs. surgical specimens) in assessing HER2 expression in patients with unresectable advanced/recurrent gastric cancer. Data were collected from gastric cancer patients who received chemotherapy at a single public hospital in Japan from 2008 to 2021. The analysis included 177 patients (109 men, 68 women) with a median age of 68.0 (21-88) years, and the primary outcome was the HER2 positivity rate. The overall HER2 positivity rate was 18.1%, with higher rates on weekdays (20.0%) compared to weekends (12.8%). Biopsies had higher positivity rates on weekdays (23.9%) but lower rates on weekends (11.1%) than surgical specimens. Significant differences were observed in formalin fixation times between weekdays and weekends for both biopsies and surgical samples. The study findings suggest that longer formalin fixation times on weekends may lead to underestimating HER2 expression, particularly in biopsies. Therefore, it is crucial to be cautious of excessive formalin fixation when collecting samples, especially during weekend biopsies.

CD47 polymorphism for predicting nivolumab benefit in patients with advanced non­small­cell lung cancer.

Immune checkpoint inhibitors (ICIs), such as nivolumab, play an essential role in non-small-cell lung cancer (NSCLC) treatment. Programmed death ligand-1 has been used as a predictive biomarker for the efficacy of ICI treatment in patients with NSCLC; however, its predictive value is considered insufficient. Therefore, there is an urgent need for better predictive biomarkers. The present study focused on the CD47 molecule, which is associated with macrophages and tumor immunity. The study aimed to investigate the association between CD47 single nucleotide polymorphism (SNP) and the therapeutic effect of nivolumab in patients with NSCLC. The CD47 SNP genotypes and clinical outcomes were retrospectively analyzed in 164 patients with NSCLC treated with nivolumab at Kyoto University Hospital (Kyoto, Japan). Patients with the G/G genotype of the CD47 SNP rs3804639 had significantly longer progression-free survival than those with the G/T or T/T genotypes [2.6 months vs. 2.1 months, hazard ratio (HR), 0.70; P=0.026]. Moreover, the G/G genotype of the CD47 SNP rs3804639 was associated with a significantly longer median overall survival than the G/T or T/T genotypes of the CD47 SNP rs3804639 (24.8 months vs. 12.0 months, HR, 0.64; P=0.021). In conclusion, CD47 polymorphism may be a novel predictive biomarker of nivolumab efficacy in patients with advanced NSCLC.

BK Polyomavirus-Associated Urothelial Carcinoma of the Bladder with a Background of BK Polyomavirus Nephropathy in a Kidney Transplant Recipient.

Renal transplant recipients are at increased risk for the development of a malignant neoplasm. Polyomavirus-associated urothelial carcinoma is a rare tumor that occurs in renal transplant recipients, with approximately 41 cases reported since 2002. It accounts for 27-31% of all post-transplant urothelial carcinomas and develops at an average of 8.5 years after transplantation. Histologically, it shows high-grade urothelial carcinoma (95.1%) with a high frequency of glandular differentiation and micropapillary structures (58.5%) and positive immunohistochemistry for polyomavirus large T antigen, p53 (92.9%), and p16 (100%). We encountered a case of BK polyomavirus (BKPyV)-associated urothelial carcinoma of the bladder diagnosed 54 months after kidney transplantation. Histologically, it was a high-grade urothelial carcinoma with micropapillary features, and immunohistochemically, it was diffusely positive for polyomavirus large T antigen, p16, and p53. BKPyV DNA and mRNA for BKPyV large T antigen have been identified in tissues using real-time polymerase chain reaction. The same sequence of the BKPyV VP1 genome hypervariable region was detected in both transplanted kidney tissue with polyomavirus nephropathy and urothelial carcinoma tissue, suggesting that polyomavirus-associated urothelial carcinoma developed in a background of persistent polyomavirus nephropathy. This case showed typical histological features and was detected and treated at an earlier stage than has been reported. It is important to keep in mind that polyomavirus-associated urothelial carcinoma can develop early after transplantation and might be associated with polyomavirus nephropathy. Because of its rapidly progressive nature, careful follow-up with urine cytology and cystoscopy is necessary. We report this case with a literature review.

Continuous Monitoring of the Hydration Behavior of Hydrophilic Matrix Tablets Using Time-Domain NMR.

Time-domain NMR (TD-NMR) was used for continuous monitoring of the hydration behavior of hydrophilic matrix tablets. The model matrix tablets comprised high molecular weight polyethylene oxide (PEO), hydroxypropyl methylcellulose (HPMC), and polyethylene glycol (PEG). The model tablets were immersed in water. Their T2 relaxation curves were acquired by TD-NMR with solid-echo sequence. A curve-fitting analysis was conducted on the acquired T2 relaxation curves to identify the NMR signals corresponding to the nongelated core remaining in the samples. The amount of nongelated core was estimated from the NMR signal intensity. The estimated values were consistent with the experiment measurement values. Next, the model tablets immersed in water were monitored continuously using TD-NMR. The difference in hydration behaviors of the HPMC and PEO matrix tablets was then characterized fully. The nongelated core of the HPMC matrix tablets disappeared more slowly than that of the PEO matrix tablets. The behavior of HPMC was significantly affected by the PEG content in the tablets. It is suggested that the TD-NMR method has potential to be utilized to evaluate the gel layer properties, upon replacement of the immersion medium: purified (nondeuterated) water is replaced with heavy (deuterated) water. Finally, drug-containing matrix tablets were tested. Diltiazem hydrochloride (a highly water-soluble drug) was employed for this experiment. Reasonable in vitro drug dissolution profiles, which were in accordance with the results from TD-NMR experiments, were observed. We concluded that TD-NMR is a powerful tool to evaluate the hydration properties of hydrophilic matrix tablets.

Development and nationwide validation of kidney graft injury markers using urinary exosomes and microvesicles (complete English translation of the Japanese version).

BACKGROUND: Non-invasive, prompt, and proper detection tools for kidney graft injuries (KGIs) are awaited to ensure graft longevity. We screened diagnostic biomarkers for KGIs following kidney transplantation using extracellular vesicles (EVs; exosomes and microvesicles) from the urine samples of patients. METHODS: One hundred and twenty-seven kidney recipients at 11 Japanese institutions were enrolled in this study; urine samples were obtained prior to protocol/episode biopsies. EVs were isolated from urine samples, and EV RNA markers were assayed using quantitative reverse transcription polymerase chain reaction. Diagnostic performance of EV RNA markers and diagnostic formulas comprising these markers were evaluated by comparison with the corresponding pathological diagnoses. RESULTS: EV CXCL9, CXCL10, and UMOD were elevated in T-cell-mediated rejection samples compared with other KGI samples, while SPNS2 was elevated in chronic antibody-mediated rejection (cABMR) samples. A diagnostic formula developed through Sparse Logistic Regression analysis using EV RNA markers allowed us to accurately (with an area under the receiver operator characteristic curve [AUC] of 0.875) distinguish cABMR from other KGI samples. EV B4GALT1 and SPNS2 were also elevated in cABMR, and a diagnostic formula using these markers was able to distinguish between cABMR and chronic calcineurin toxicity accurately (AUC 0.886). In interstitial fibrosis and tubular atrophy (IFTA) urine samples and those with high Banff chronicity score sums (BChS), POTEM levels may reflect disease severity, and diagnostic formulas using POTEM detected IFTA (AUC 0.830) and high BChS (AUC 0.850). CONCLUSIONS: KGIs could be diagnosed with urinary EV mRNA analysis with relatively high accuracy.

Exacerbation of Intimal Fibrosis and Endarteritis in a Kidney Transplant Recipient with Chronic Active Antibody-Mediated Rejection and COVID-19: A Case Report.

Kidney transplant recipients are immunocompromised hosts at risk for comorbidity and mortality due to infection. Currently, there are no established guidelines for the management of immunosuppressed transplant recipients with coronavirus disease 2019 (COVID-19). The impact of COVID-19 and its therapeutic management on chronic active antibody-mediated rejection (CAAMR) are still unclear. Here, we report a case of CAAMR exacerbation with endarteritis and intimal fibrosis after COVID-19. A 41-year-old female kidney transplant recipient with CAAMR was admitted to a local hospital with moderately severe COVID-19. Her doses of tacrolimus and mycophenolate mofetil were reduced, and she was administered methylprednisolone pulse and antiviral drugs. This resulted in a good clinical course and she was discharged in 15 days. During and after hospitalization, the immunosuppressants were gradually returned to the baseline levels. However, about 1.5 months after discharge, the serum creatinine level became elevated. An indication kidney biopsy showed CAAMR with intimal fibrosis and endarteritis in all interlobular arteries. An increase of immunosuppressant led to a decrease of the serum creatinine level. Factors contributing to CAAMR with intimal fibrosis and endarteritis may include (1) insufficient immunosuppression due to changes in the levels of immunosuppressive; (2) overlap with endothelial cell injury caused by COVID-19, and (3) an immune-activated state associated with COVID-19. COVID-19 is a life-threatening disease that can result in unexpected changes in immunological status. Possible allograft rejection should be carefully managed in such patients.

Mechanism and treatment for chronic antibody-mediated rejection in kidney transplant recipients.

Chronic antibody-mediated rejection of kidney transplantation is a major cause of late-stage graft loss. Donor-specific antibodies are the main cause of antibody-mediated rejection; in particular, de novo donor-specific antibodies are a risk factor for chronic active antibody-mediated rejection. The level of de novo donor-specific antibodies tends to increase with time throughout long-term graft survival. Donor-specific antibodies induce humoral rejection through complement activation, which results in tissue injury and coagulation. Additionally, complement activation promotes the migration of inflammatory cells through the innate immune response, causing endothelial injury. This inflammatory response may cause persistent glomerulitis and peritubular capillaritis, leading to fixed pathological lesions that impair graft function. No treatment has been established for chronic antibody-mediated rejection, a condition in which antibody-mediated rejection becomes irreversible. Thus, antibody-mediated rejection must be detected and treated while it is still reversible. In this review, we discuss the development of de novo donor-specific antibodies and the mechanisms leading to chronic antibody-mediated rejection and summarize the current treatment options and the latest biomarkers for detecting chronic antibody-mediated rejection at an earlier stage.

CD47 blockade ameliorates autoimmune vasculitis via efferocytosis of neutrophil extracellular traps.

Neutrophil extracellular trap (NET) formation contributes to immune defense and is a distinct form of cell death. Excessive NET formation is found in patients with anti-neutrophil cytoplasmic antibody-associated (ANCA-associated) vasculitis (AAV), contributing to disease progression. The clearance of dead cells by macrophages, a process known as efferocytosis, is regulated by the CD47-mediated "don't eat me" signal. Hence, we hypothesized that pathogenic NETs in AAV escape from efferocytosis via the CD47 signaling pathway, resulting in the development of necrotizing vasculitis. Immunostaining for CD47 in human renal tissues revealed high CD47 expression in crescentic glomerular lesions of patients with AAV. In ex vivo studies, ANCA-induced netting neutrophils increased the expression of CD47 with the reduction of efferocytosis. After efferocytosis, macrophages displayed proinflammatory phenotypes. The blockade of CD47 in spontaneous crescentic glomerulonephritis-forming/Kinjoh (SCG/Kj) mice ameliorated renal disease and reduced myeloperoxidase-ANCA (MPO-ANCA) titers with a reduction in NET formation. Thus, CD47 blockade would protect against developing glomerulonephritis in AAV via restored efferocytosis of ANCA-induced NETs.

Key Points of the Banff 2019 Classification of Renal Allograft Pathology.

The Banff 2019 Kidney Meeting Report was published following the 15th Banff Conference for Allograft Pathology, which was held in Pittsburgh on September 23-28, 2019. The diagnosis of renal transplant rejection based on the Banff 2019 classification is globally utilized. The Banff 2019 classification of renal allograft pathology includes several changes. This mini-review summarizes the key points and issues of the Banff 2019 classification. The criteria for borderline change have been reverted to ≥ i1, the t-IFTA score has been incorporated into the classification, the histological classification of polyoma virus nephropathy has been adopted, and a category was added for chronic (inactive) antibody-mediated rejection. In addition, whether the spread is diffuse or focal should now be noted in the presence of peritubular capillaritis. One of the issues with the Banff 2019 classification is the continuing lack of clarity regarding the definition of the t score, which is used to evaluate tubulitis not only in non-scarred areas but also in moderately atrophic tubules that are assumed to be present in scarred areas, which constitutes a contradiction in the definition.

Sternoclavicular Septic Arthritis Due to Invasive Pneumococcal Infection After Type A Influenza Virus Infection.

A 24-year-old female patient who had a type A influenza virus infection prior to admission visited our hospital complaining of a fever and right sternoclavicular pain. Blood culture was positive for penicillin-sensitive Streptococcus pneumoniae (pneumococcus). Magnetic resonance imaging of the right sternoclavicular joint (SCJ) showed a high signal intensity area on the diffusion-weighted images. Consequently, the patient was diagnosed with septic arthritis due to invasive pneumococcus. When a patient complains of gradually increasing chest pain after an influenza virus infection, SCJ septic arthritis should be considered in the differential diagnosis.

Optic Nerve Injury Enhanced Mitochondrial Fission and Increased Mitochondrial Density without Altering the Uniform Mitochondrial Distribution in the Unmyelinated Axons of Retinal Ganglion Cells in a Mouse Model.

Glaucomatous optic neuropathy (GON), a major cause of blindness, is characterized by the loss of retinal ganglion cells (RGCs) and the degeneration of their axons. Mitochondria are deeply involved in maintaining the health of RGCs and their axons. Therefore, lots of attempts have been made to develop diagnostic tools and therapies targeting mitochondria. Recently, we reported that mitochondria are uniformly distributed in the unmyelinated axons of RGCs, possibly owing to the ATP gradient. Thus, using transgenic mice expressing yellow fluorescent protein targeting mitochondria exclusively in RGCs within the retina, we assessed the alteration of mitochondrial distributions induced by optic nerve crush (ONC) via in vitro flat-mount retinal sections and in vivo fundus images captured with a confocal scanning ophthalmoscope. We observed that the mitochondrial distribution in the unmyelinated axons of survived RGCs after ONC remained uniform, although their density increased. Furthermore, via in vitro analysis, we discovered that the mitochondrial size is attenuated following ONC. These results suggest that ONC induces mitochondrial fission without disrupting the uniform mitochondrial distribution, possibly preventing axonal degeneration and apoptosis. The in vivo visualization system of axonal mitochondria in RGCs may be applicable in the detection of the progression of GON in animal studies and potentially in humans.

A Case of Kidney Transplantation from a Deceased Donor with Acute Kidney Injury due to Rhabdomyolysis.

Acute kidney injury (AKI) due to rhabdomyolysis occurs because of renal ischemia or acute tubular necrosis due to the deposition of myoglobin casts in the renal tubules. Donors with AKI due to rhabdomyolysis are not contraindication for transplantation. However, the dark red kidney raises concerns about renal hypofunction or primary nonfunction after transplantation. We report the case of a 34-year-old man with a 15-year history of hemodialysis for chronic renal failure due to congenital anomalies of the kidney and urinary tract. The patient received a renal allograft from a young woman who suffered cardiac death. The serum creatinine (sCre) level of the donor at the time of transport was 0.6 mg/dL, and renal ultrasonography revealed no abnormalities in renal morphology or blood flow. Her serum creatinine kinase level increased to 57,000 IU/L 58 h after femoral artery cannulation and sCre level worsened to 1.4 mg/dL, suggesting AKI due to rhabdomyolysis. However, since the urine output of the donor was maintained, the sCre elevation was thought to be nonproblematic. The allograft had a dark red appearance at the time of procurement. The perfusion of the isolated kidney was good, but the dark red color did not improve. A 0-h biopsy showed flattening of the renal tubular epithelium and absence of the brush border and myoglobin casts in 30% of the renal tubules. Rhabdomyolysis-related tubular damage was diagnosed. Hemodialysis was discontinued on postoperative day 14. Twenty-four days after the operation, the transplanted kidney function progressed favorably (sCre 1.18 mg/dL), and the patient was discharged. Protocol biopsy 1 month after transplantation showed disappearance of myoglobin casts and improvement in renal tubular epithelial damage. The patient's sCre level was approximately 1.0 mg/dL 24 months after transplantation, and he is doing well without complications.

Oral Supplementation with Maca Improves Social Recognition Deficits in the Valproic Acid Animal Model of Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a congenital, lifelong neurodevelopmental disorder whose main symptom is impaired social communication and interaction. However, no drug can treat social deficits in patients with ASD, and treatments to alleviate social behavioral deficits are sorely needed. Here, we examined the effect of oral supplementation of maca (Lepidium meyenii) on social deficits of in utero-exposed valproic acid (VPA) mice, widely used as an ASD model. Although maca is widely consumed as a fertility enhancer and aphrodisiac, it possesses multiple beneficial activities. Additionally, it benefits learning and memory in experimental animal models. Therefore, the effect of maca supplementation on the social behavioral deficit of VPA mice was assessed using a social interaction test, a three-stage open field test, and a five-trial social memory test. The oral supplementation of maca attenuated social interaction behavior deficit and social memory impairment. The number of c-Fos-positive cells and the percentage of c-Fos-positive oxytocin neurons increased in supraoptic and paraventricular neurons of maca-treated VPA mice. These results reveal for the first time that maca is beneficial to social memory and that it restores social recognition impairments by augmenting the oxytocinergic neuronal pathways, which play an essential role in diverse social behaviors.